The goal of this video is to demonstrate the method for the isolation and identification of RNAs and protein components of ribonucleoprotein complexes from cell extracts using immunoprecipitation methods. Hello, my name is Garrett Dom and I'm a graduate student in the Molecular and Microbiology and immunology department at the University of Missouri studying under Dr.Eli sso. From the advancement in high throughput sequencing and efficient microarray analysis, global gene expression and analysis has become an easy and readily available form of data collection in many research and disease models.
However, study state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the interactions of RNA binding proteins.
Post transcriptional regulation affects mRNA localization, stability and translation by forming a rib nucleo protein complex with target mRNAs. Identifying these unknown Denovo mRNA targets from cellular extracts in this complex is pivotal to understanding the mechanisms and functions of the RNA binding protein and their resulting effect on protein output. This protocol outlines a method termed ribo protein, immuno immunoprecipitation, or rip, which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex.
Before starting the experiment, it is critical to have all reagents, containers and utensils. RNAs free treat glassware with R nase inhibitor such as RNA ZAP from ambion, followed by rinsing with dpci treated water, ensure that all reagents are confirmed as RNAs free. The first step of the procedure is to harvest the rib nucleo protein complex from the cellular lysate harvest exponentially growing tissue cells to produce between two to five milligrams of total protein for each sample used, usually two P one 50 culture dishes are sufficient.
Here we are harvesting MB 2 31 and MC seven human breast cancer cell lines and investigating the ribon nucleo interactions of the RNA binding protein. Who are it is important to note for each RNA binding protein being investigated, total cellular number and protein amounts must be optimized to maximize appropriate target mRNA and RNA binding protein interactions. Pellet cells via centrifugation at 1000 RPM for approximately eight to 10 minutes at four degrees Celsius, then wash three times with ice cold PBS After the final wash, gently flick the bottom of the tube and resuspend cell pellet in equal volumes of prepared poly somal lysis buffer with RNAs and protease inhibitors.
It's recommended to measure out the exact volume of the cell pellet gently pipette mixture to break apart clumps of cells and incubate lysate on ice for five minutes. Centrifuge at 13, 000 G for 20 minutes at four degrees Celsius to clear the lysate of debris immediately transfer cleared supernatant to pre chilled RNAs. Free micro centrifuge tubes keep on ice and store at minus 80 degrees Celsius.
This lysate can be stored for up to six months at minus 80 degrees Celsius. Avoid repeated freeze thaw cycles as this can lead to protein and or mRNA degradation. It is best to do the IP immediately.
Do a quick quantitate the concentration of protein and the lysate using standard Bradford protein assay. Next, we will need to prepare the materials for the isolation of our specific protein of interest. First pre swell protein.
A Sphero beads overnight in N NT two buffer supplemented with 5%BSA store at four degrees Celsius. Use NT two buffer at a four to one ratio with PAS beads. Long-term storage of up to several months at four degrees Celsius is possible.
When supplemented with 0.1%sodium azi before use, remove excess NT two buffer so that the final beads to buffer ratio is one-to-one. Using 1.5 milliliter RNA free micro centrifuge tubes, remove 100 microliters of SRO speed slurry and add 30 micrograms of antibody for each individual IP reaction. For example, in our experiment we use a who are antibody, which is a mouse IgG one antibody.
Therefore our control antibody is also IgG one. Next, add 100 to 200 microliters of NT two buffer to antibody bead mix. Additionally, an isotype matched antibody or whole normal serum from same species should be used in parallel as an antibody control against background RNA.
Add appropriate antibody to bead mix and incubate overnight tumbling in over end at four degrees Celsius. Prep antibody coated beads immediately before use by washing with one milliliter of ice cold NT two buffer five times wash beads by centrifugation at 13, 000 G for one to two minutes at four degrees. Carefully remove supernatant with hand pipetter or aspirator.
This washing helps remove unbound antibody as well as RNAs contaminants from antibody mixture. Once the final wash has been completed, resus suspend the beads and 700 microliters of ice cold T two buffer, followed by treatment with various RNAs inhibitors to protect the target mRNAs, including 10 microliters of RNAs, out 10 microliters of 100 millimolar DTT and 15 microliters of EDTA. Bring volume to 900 microliters with NT two buffer.
Next, we will immuno precipitate the target complex. We strongly recommend using a pre-clear step to reduce background signal in your RNA analysis after rip procedure pre-clear with 15 micrograms of isotype control for 30 minutes at four degrees Celsius. Add 50 microliters of pres swollen PAS beads non coated with antibody.
Incubate 30 minutes at four degrees Celsius with rotation and over in centrifuge at 10, 000 G at four degrees Celsius. Two pellet. Save the super natin for ip.
Next, add 100 microliters of isolated cleared lysate with approximately two to five milligrams of protein to prepared antibody mix. Diluting lysate will help to reduce background wrap tube and paraform to ensure tight ceiling and incubate at four degrees Celsius for four hours tumbling and over in next pellet beads at 5, 000 G for five minutes at four degrees Celsius and save supernat for potential analysis by storing at minus 80 degrees Celsius as described previously. Wash beads five times with one milliliter of ice cold and T two buffer and centrifugation.
Then remove super natin with hand pipetter or an aspirator. More stringent methods may be used in order to reduce background by supplementing NT two buffer with sodium deoxy coate, an uria or SDS. Keep samples on ice as much as possible and work quickly to minimize degradation of target mRNA.
Finally, we will use DNAs and proteinase K treatments followed by RNA precipitation to isolate the ribon nuclear protein RNAs after washing resuspend beads with 100 microliters of N NT two buffer supplemented with five microliters of RNAs free DNAs one. Keep samples at 37 degrees Celsius for five to 10 minutes. Add one milliliter of N NT two buffer and spin at 5, 000 G for five minutes.
Discard SUPERNAT Reese spin protein a SROs pellet and 100 microliters of NT two buffer with five microliters of proteinase K at 10 milligrams per milliliter and one microliter of 10%SDS incubate the resuspended bead mixture for 30 minutes in a 55 degree Celsius water bath gently flicking every 10 minutes. Proteinase K will aid in the release of the rib nucleo protein components. Pellet beads.
Add 5, 000 G for five minutes at room temperature, collect the super name, which should be roughly 100 microliters in volume. Next two beads, add 200 microliters of NT two buffer centrifuge at 5, 000 G for two minutes. Collect approximately 200 microliters of super natin and discard beads.
Combine super natin and add 300 microliters of lower layer acid phenol vortex. One minute at room temperature and centrifuge max speed at room temperature for one minute. Collect 250 microliters of upper layer.
Be very careful to not disturb the interface as this will contaminate. RNA sample add 25 microliters of sodium acetate at pH of 5.2 625 microliters of chilled, 100%ethanol and five microliters of glyco blue mix. Well store overnight at minus 20 degrees Celsius.
Additionally to ensure proper recovery of RNA. The addition of glyco blue will increase the visibility of the RNA pellet by acting as a carrier molecule the following day, mixed tubes by inverting three to five times, then spin at 12, 000 G at four degrees Celsius for 30 minutes and discard the super natin. Add one milliliter of chilled, 70%ethanol to the blue pellet and mixed by inversion or vortexing centrifuge.
Sample at 12, 000 G at four degrees Celsius for two minutes. Discard supernat and centrifuge at 12, 000 G for one minute at four degrees Celsius. Remove any residual, 70%ethanol with a pipet and air dry pellet.
Add room temperature for five minutes. Re spend in 20 to 40 microliters of RNAs, DNA free water. The sample is now ready for quantitation by NanoDrop spectrometry and further downstream applications such as quantitative real-time PCR or microarray analysis after RNA isolation, microarray and quantitative real-time PCR were performed to reveal the mRNA targets of the RNA binding protein who are, and their resulting enrichment from the immunoprecipitation.
Confirmation by western blot reveals clean isolation of who are protein. Quantitative PCR demonstrates the fold enrichment of the mRNA target beta actin compared with IgG one controls. While microarray analysis reveals unique genetic profiles between MC seven and MB 2 31, breast cancer cell lines overall rib nucleo protein immunoprecipitation has been established as an excellent tool used to isolate and study the interactions between RNA binding proteins and their MR.A targets by our group, as well as many other research groups, though sensitive in nature and practice, proper execution of this procedure will yield the isolation of these rib nucleo protein complexes, which until recently have been inaccessible for discovery and analysis.
